Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (αβ) 2 dimer

Abstract

We describe and compare the main kinetic characteristics of the (αβ) 2 form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high ( K 0.5=4 μM) and low ( K 0.5=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate ( K 0.5=0.5 mM) and a lower hydrolysis rate ( V M=42 U/mg). Stimulation of ATPase activity by K + ( K 0.5=0.63 mM), Na + ( K 0.5=11 mM) and Mg 2+ ( K 0.5=0.60 mM) all showed V M's of approximately 600 U/mg and negative cooperativity. K + ( K 0.5=0.69 mM) and Mg 2+ ( K 0.5=0.57 mM) also stimulated PNPPase activity of the (αβ) 2 form. Ouabain ( K 0.5=0.01 μM and K 0.5=0.1 mM) and orthovanadate ( K 0.5=0.06 μM) completely inhibited the ATPase activity of the (αβ) 2 form. The kinetic characteristics obtained constitute reference values for diprotomeric (αβ) 2-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.